In vitro proteolysis of ovine lens crystallins by calpain II : A dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours

Abstract

A cataract is any opacity of the lens caused by unregulated protein aggregation. Cataracts are the major cause of human blindness worldwide and it is thought that calpain-induced degradation of the major lens proteins, the crystallins, is an important component in many types of cataract.. At Lincoln University, a heritable cataract in sheep is being studied. The aim of this project was to set up an in vitro system to investigate the ovine crystallin degradation products produced by calpain II-induced proteolysis to enable comparison with crystallin degradation during cataract formation. Ovine lens α-, β-and γ- crystallins and calpain II were purified from sheep lens and lung respectively. Calpain II separation was achieved using ammonium sulphate precipitation, hydrophobic chromatography and ion exchange chromatography, while the crystallins were purified using ion exchange and gel filtration chromatography. Calpain II was incubated separately with α-, β-and γ- crystallin and the resulting products were analysed by SDS PAGE. Calpain II was found to cause proteolytic degradation of α- β- crystallins but not γ- crystallins. Degradation of the double α--crystallin bands of 20kDa and 25Da produced three new bands at 22kDa, 19kDa and 18kDa on an SDS PAGE gel. β-Crystallin was represented on SDS PAGE gels as five bands of 31kDa, 29kDa, 27kDa, 26kDa and 24kDa Calpain II induced degradation yielded one new visible band at 30kDa and broadened the 26kDa band, presumably by superimposition. The 31kDa and 29kDa bands disappeared entirely, while the 27kDa band thinned considerably. The bottom 24kDa β-crystallin band appeared to remain unchanged. This project therefore provided evidence of crystallin-specific degradation by calpain II, but further work is required to satisfactorily characterise the cleavage products for comparison with the in vivo ovine cataract model.